Docosahexaenoic acid (22:6n-3; DHA) is an abundant component in the brain phospholipids. It plays an important role in prenatal brain development and maintenance of normal brain function. The brain DHA deficiency can reduce normal levels of neural membrane DHA molecular species of phospholipids, leading to markedly influencing optimal learning and memory [Fedorova, et. al., An n-3 fatty acid deficiency impairs rat spatial learning in the Barnes maze, Behav. Neurosci., 123, 196 (2009)] and causing neuronal apoptosis [Kim, et. al., Inhibition of neuronal apoptosis by polyunsaturated fatty acids, J. Mol. Neurosci. 16, 223 (2001)].
It has been demonstrated that the maintenance of normal levels of neural membrane DHA phosphatidylserine and DHA plasmalogen phosphatidylethanolamine species [Favrelere, et. al., Age-related changes in ethanolamine phospholipid fatty acid levels in rat frontal cortex and hippocampus, Neurobiol. Aging 21, 653 (2001); McGahon, et. al., Age-related changes in synaptic function: analysis of the effect of dietary supplementation with omega-3 fatty acids, Neuroscience 94, 305 (1999)] is essential for keeping normal membrane function [Glomset, Role of docosahexaenoic acid in neuronal plasma membrane. Sci. STKE, page 6, (2006); Salem et. al., Mechanisms of action of docosahexaenoic acid in the nervous system, Lipids 36, 945 (2001)]. Continuous supply of DHA into the brain and unique metabolism of DHA in relation to its incorporation into neural membrane phospholipids plays an important role in maintaining both neural membrane fluidity and gap junction coupling capacity, in order to keep normal expression of neurotrophin receptors and effective retrograde transport of the NGF-neurotrophin receptor complexes from cerebral cortex and hippocampus to basal forebrain [Kim, Novel metabolism of docosahexaenoic acid in neural cells, J. Biol. Chem. 282, 18661 (2007); Champeol-Potokar, et. al., Docosahexaenoic acid (22:6n-3) enrichment of membrane phospholipids increases gap junction coupling capacity in cultured astrocytes, Euro. J. Neurosci. 24, 3084 (2006); Farooqui, et, al., Biochemical aspects of neurodegeneration in human brain: involvement of neural membrane phospholipids and phospholipase A2, Neurochemical Res. 29, 1961 (2004)] in order to inhibit neuronal apoptosis because an important neurotransmitter acetylcholine is synthesized mainly in basal forebrain cholinergic neurons [Terry and Buccafusco, The cholinergic hypothesis of age and Alzheimer's disease-related cognitive deficits: Recent challenges and their implications for novel drug development. The Journal of Pharmacology and Experimental Therapeutics, 306, 821 (2003); Auld, et. al., Nerve growth factor induces prolonged acetylcholine release from cultured basal forebrain neurons: differentiation between neuromodulatory and neurotrophinc influences, J. Neurosci. 21, 3375 (2001)]. It has been reported that acetylcholine synthesis, choline acetyltransferase activity and expression of p-75 neurotrophin receptor in patients with Alzheimer's disease have been found to be markedly reduced at least 40%, compared with controls [Sims, et. al., Presynaptic cholinergic dysfunction in patients with dementia, J. Neurochem. 40, 503 (2006); Mufson, et al., Loss of basal forebrain P75NTR immunoreactivity in subjects with mild cognitive impairment and Alzheimer's disease. J. Comparative Neurology 443, 136 (2002)].
The following scheme shows the pathway of biosynthesis and metabolism of an important neurotransmitter acetylcholine in neurons:

The mechanism of the current drugs for treatment of age-dependent cholinergic dysfunction related neurodegenerative disorders is to inhibit the activity of acetylcholinesterase, in order to decrease further degradation of acetylcholine in the brain [Grutzendler and Morris, Cholinesterase inhibitors for Alzheimer's disease, Drugs 61, 41 (2001)], rather than to promote neuronal survival. Because age-dependent neurodegenerative diseases are a progressive disorder, the cure becomes much more difficult or even impossible if the prevention and treatment start at a later stage of the diseases. However, an ideal drug used for such disorders should enable to both simultaneously delay or halt the underlying pathological process and improve memory and other clinical deficits.
The study further indicates that neuronal apoptosis under adverse conditions can be prevented by DHA enrichment in a phosphatidylserine (PS)—dependent manner, and depletion of DHA from neuronal tissues can influence biosynthesis and accumulation of PS [Kim, et. al., Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species, Biochemistry, 43, 1030 (2004)]. Furthermore, it is also important to point out that the provision of docosapentaenoic acid (22:5n-6; DPA) in place of DHA is sufficient neither for fully supporting PS accumulation nor for neuronal survival [Kim, et. al., Effects of docosahexaenoic acid on neuronal apoptosis, Lipids 38, 453 (2003); Lim et. al., An extraordinary degree of structural specificity is required in neural phospholipids for optimal brain function: n-6 docosapentaenoic acid substitution for docosahexaenoic acid leads to a loss in spatial task performance, J. Neurochem. 95, 848 (2007)].
It is clear that DHA positively modulates biosynthesis and accumulation of neural membrane DHA PS species, promoting neuronal survival. Rising the level of PS by DHA enrichment can be observed only in neural cells, representing a unique mechanism for expending DHA PS and PS pools in mammalian neurons, in order to inhibit neuronal apoptosis [Hamilton, et. al., n-3 fatty acid deficiency decreases phosphatidylserine accumulation selectively in neuronal tissues, Lipids, 35, 863 (2000); Guo, et. al., Neuronal specific increase of phosphatidylserine by docosahexaenoic acid, J. Mol. Neurosci. 33, 67 (2007)].
In summary, a DHA deficient diet can cause brain DHA deficiency, especially in aging adults. Effective supply of DHA to brain tissues in order to keep normal levels of DHA or to reverse abnormal levels of neural membrane DHA PS is of vital importance in the prevention and treatment of age-dependent neurodegenerative disorders [Kim, et. al., Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3). J. Biol. Chem. 275, 35215 (2000); Kim, Biochemical and biological functions of docosahexaenoic acid in the nervous system: modulation by ethanol, Chem. Phys Lipids, 153, 34 (2008); Editor's Choice, DHA Increases PS to Promote Neuronal Survival, Sci. STKE, 2005, p286 (2005)].
Because it cannot be synthesized in the brain, DHA has to be supplied entirely from the diet and is then delivered into the brain by plasma [Spector, Plasma free fatty acid and lipoproteins as sources of polyunsaturated fatty acids for the brain. J. Mol. Neurosci. 16, 159 (2001)]. Unlike other tissues, the brain uptake of DHA needs to overcome the blood-brain barrier (BBB). However, to further understand the mechanism by which DHA phospholipid carriers pass across the BBB is important in preparing mechanism-based DHA phospholipid transporters.
Generally, DHA can be delivered into the brain in forms of both non-esterified DHA and phospholipids. The DHA carried by lysophospholipids is preferred transporters to pass across the BBB [Chen and Subbaiah, Phospholipid and fatty acid specificity of endothelial lipase: potential role of the enzyme in the delivery of docosahexaenoic Acid (DHA) to tissues, Biochim. Biophys. Acta, 171, 1319 (2007); Lagarde et. al., Lysophosphatidylcholine as preferred carrier form of docosahexaenoic acid to the brain, J. Mol. Neurosci. 16, 201 (2001); Thies et. al., Unsaturated fatty acids esterified in 2-acyl-1-lysophosphatidylcholine bound to albumin are more efficiently taken up by the young rat brain than the non-esterified form, J. Neurochem. 59, 1110 (1992)] because the incorporation of DHA into the brain is approximately 10-fold higher from enzyme-catalyzed 2-DHA lysophospholipids than from non-esterified DHA at various times of analyses [Lagarde, Docosahexaenoic acid: Neutrient and precursor of bioactive lipids, Eur. J. Lipid Sci. Technol. 110, 673 (2008)].
For lipids based brain DHA transporters, use of a composition that consists of highly enriched DHA-containing lipid species is a must. Orally administrated DHA triglyceride carriers, which are present in current supplements including fish, algae and krill oils, have shown benefits for the human health, but it is still questionable whether these lipid mixtures are qualified as effective DHA transporters in the delivery of DHA into the brain, especially aged brain [Arendash, et. al., A diet high in omega-3 fatty acids does not improve or protect cognitive performance in Alzheimer's transgenic mice, Neuroscience, 149: 286 (2007); Freund-Levi et al., co-3 fatty acid treatment in 174 patients with mild to moderate Alzheimer's disease: Omeg AD study, Arch Neurol. 63; 1402 (2006)]. Because DHA lipid species metabolite differently from others, it is hard to further understand pharmacological and nutritional functions of those DHA lipid species in a mixture form. [Hossain, et. al, Docosahexaenoic acid and eicosapentaenoic acid-enriched phosphatidylcholine liposomes enhance the permeability, transportation and uptake of phospholipids in Caco-2 cells. Molecular and Cellular Biochemistry, 285, 155 (2006); Chen and Subbaiah, Phospholipid and fatty acid specificity of endothelial lipase: potential role of the enzyme in the delivery of docosahexaenoic Acid (DHA) to tissues, Biochim. Biophys. Acta, 171, 1319 (2007)]. Although the ethyl DHA/EPA drug (over 80% purity) may alleviate coronary atherosclerosis, it may not be qualified as brain DHA transporters to overcome the blood-brain barrier (BBB).
On the other hand, the capacity of the brain to convert α-linolenic acid (18:3n-3; ALA) or eicosapentaenoic acid (20:5n-3; EPA) to DHA is limited [Igarashi et. al., Docosahexaenoic acid synthesis from alpha-linolenic acid by rat brain is unaffected by dietary n-3 PUFA deprivation, J. Lipid Res. 48: 1150 (2007)]. For example, converting ALA and EPA to DHA involves more than single desaturation and elongation step, and yields of the DHA formed by this route is very low [Brenna, et. al., alpha-Linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans, Prostaglandins Leukot. Essent. Fatty Acids, 80, 85 (2009)]. Due to structural specificity of DHA for brain requirements, the study strongly suggests that a direct supply of DHA to the brain is required against neuronal apoptosis [Kim, et. al., Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3), J. Biol. Chem. 275, 35215 (2000)].
Because brain DHA deficiency can cause brain function disorders, particular during periods of brain development and aging, the methods of using various compositions of lipid nutrients and potential drugs have been applied for enhancing levels of DHA in the brain, in order to improve general brain function.
U.S. Pat. No. 5,869,530 discloses a method of using phospholipids as dietary supplements for improving general brain function. Mixtures of phospholipids including phosphatidylcholine and phosphatidylethanolamine are extracted from chicken egg yolk, but DHA molecular species obtained from this natural resource are absent.
U.S. Pat. No. 5,716,614 discloses a method of transporting DHA into the brain by EPA and DHA aminophospholipids-conjugated polycationic carriers (e.g. poly-lysine and poly-arginine or poly-ornithine) rather than highly pure DHA aminophospholipids, in order to improve function of mammalian brain.
Japanese patent 06256179 discloses the method for preparing 1,2-polyunsaturated fatty acids—3-phosphorycholine, or 3-phosphorylethanolmine, or 3-phosphorylserine, or 3-phosphorylinositol for improving learning ability and for treating senile dementia. However, Japanese patent 06256179 does not disclose highly enriched 1-acyl chains/2-DHA—containing molecular species of highly pure phospholipids for promoting survival of aged basal forebrain cholinergic dysfunction related neurodegenerative disorders.
Japanese 06279311 discloses the method of using a mixture of polyunsaturated fatty acids—containing phosphatidylserine species for treatment of senile dementia, especially Alzheimer's disease. However, the said compositions do not comprise a highly enriched 1-acyl chains/2-DHA—containing molecular species of highly pure phosphatidylserine, as well as highly pure phosphatidylethanolamine and highly pure phosphatidyl-monomethylethanolamine, for promoting survival of aged basal forebrain cholinergic neurons to prevent and treat age-related neurodegenerative diseases.
U.S. Pat. No. 6,964,969 discloses a method of treating impaired or deteriorating neurological function using a mixture of n-3/n-6 fatty acids and vitamins.
U.S. Pat. No. 5,668,117 discloses a method of treating age and Alzheimer's disease by administrating Vitamins. Similar effects for treatment of age and Alzheimer's disease using dehydroepiandrosterone has been also disclosed in U.S. Pat. No. 4,812,447.
WO patent 2007/073178 discloses a method of using compositions comprising DHA, proteins and manganese for improving membrane composition.
WO patent 1997/39759 discloses the methods for the preparation of 1,2-DHA-containing phosphatidylcholine species for the treatment of bipolar disorders. However, said compositions do not comprise highly enriched 2-DHA—containing molecular species of highly pure phosphatidylserine, or highly pure phosphatidylethanolamine or/and highly pure phosphatidyl-monomethylethanolamine.
WO patent 2005/051091 discloses a method of developing cognitive and vision functions of infants and children using a mixture of glycerophospholipid in combination with sphingomyelin or cholesterol.
Other published patents also documented methods of using mixtures of phospholipids, sphingomyelins and n-3 and n-6 fatty acids for the treatment of (1) a wide range of diseases [EP patent 1279400], (2) multiple traumata, burns, infections, and chronic inflammatory disease [EP patent 0311091], (3) hepatic cirrhosis and diarrhea, and (4) cancer diseases [EP patent 1426053].
From above published results, it is clear to see that the prior art does not disclose using highly pure 1-acyl chains/2-DHA aminophospholipids for promoting survival of aged basal forebrain cholinergic neurons.
The research paper and university study also reported the methods of using polyunsaturated fatty acids, such as DHA against excitotoxic brain damage of infant rats [Hogyer, et. al., Neuroprotective effect of developmental docosahexaenoic acid supplement against excitotoxic brain damage in infant rats, Neuroscience, 119, 999 (2003)]. The positive effect of long-chain polyunsaturated fatty acids on brain function in newborn and aged rats has been shown as well. But the methods of using highly pure 1-acyl chains/2-DHA aminophospholipids to promote survival of basal forebrain cholinergic neurons have not been claimed in the studies.
U.S. Pat. No. 5,654,290 discloses the methods of using polyunsaturated fatty acids based drugs, which include highly pure 2-DHA triglycerides, highly pure 1-short acyl chains/2-DHA PC species, and highly pure 2-DHA lysoPC species, to treat brain disorders. However, highly pure 1-acyl chains/2-DHA PE, highly pure 1-acyl chains/2-DHA PMME and highly pure 1-acyl chains/2-DHA PS are not disclosed in U.S. Pat. No. 5,654,290. Further, the patent does not disclose inhibiting neuronal apoptosis or treating basal forebrain cholinergic dysfunction related neurodegenerative disorders.
A method of using 1-acyl chains/2-DHA phosphatidylserine (PS) species, which is extracted from bovine cortex, as the first DHA phospholipid based drug in Europe to alleviate and treat Alzheimer's disease has been documented [Amaducci, et al., Phosphatidylserine in the treatment of Alzheimer's disease: Results of a multicenter study. Psychopharmacology Bulletin 24, 130 (1988); Crook, et al., Effect of phosphatidylserine in age-associated memory impairment. Neurology, 41, 644 (1991); Effect of phosphatidylserine in Alzheimer's disease, Psychopharmacol. Bulletin 28, 61 (1992); Pepeu, et al., A review of phosphatidylserine pharmaceutical and clinical effects: Is phosphatidylserine a drug for aging brain? Pharmacology Research, 33, 51 (1996)]. Although the purity of the bovine PS drug was over 80%, the percentage of DHA molecular species in the PS drug was about 10% [Chen and Li, Comparison of molecular species of various transphosphatidylated soy-phosphatidylserine with bovine cortex PS by mass spectrometry. Chem. Phys. Lipids, 152, 46 (2008)]. Based on previously used clinical dosage of bovine cortex PS drug (100-600 mg/day), it took at least 60 days to meet the expected effects because the actual amount of the 2-DHA species intake was in the range of 10-60 mg/daily only. The safety of oral administration of phospholipids including PS up to 600 mg/daily has been confirmed [Jorissen, et. al., Safety of soy-derived phosphatidylserine in elderly people, Nutritional Neurosci., 5, 337 (2002)].
However, the risk of bovine spongiform encephalopathy (Mad Cow Disease) made use of the PS, the first DHA phospholipid based drug, potentially unsafe. Although methods of using alternatives of DHA PS species mixtures, made by transphosphatidylation of squid skin PC [Hosokawa, et. al., Conversion to docosahexaenoic acid-containing phosphatidylserine from squid skin lecithin by phospholipase D-mediated transphosphatidylation. J. Agric. Food. Chem. 48, 4550 (2000)] and fish liver PC [Chen and Li, Comparison of molecular species of various transphosphatidylated soy-phosphatidylserine with bovine cortex PS by mass spectrometry. Chem. Phys. Lipids, 152, 46 (2008)] to improve learning ability of DHA deficient mice have been reported[http://www.issfal.org.uk/index.php?option=com_content&task=view&id=55 &Itemid=8 7%20#CS6], the maximal percentage of DHA species in the alternatives is approximately 45-55%. The method of using highly enriched 1-acyl chains/2-DHA molecular species (over 70% in the species mixture) of highly pure phosphatidylserine, highly pure phosphatidylethanolamine and highly pure phosphatidyl-monoethanolamine (more than 90% purity) for the purpose has never been reported.
For the preparation of natural source-based highly enriched 1-acyl chains/2-DHA-containing molecular species of highly pure phosphatidylserine (PS), the transphosphatidylation of highly enriched 1-acyl chain/2-DHA—containing molecular species of phosphatidylcholine [Hosokawa, et. al., Preparation of therapeutic phospholipids through porcine pancreatic phospholipase A2-mediated esterification and lipozyme-mediated acidolysis. J. Am. Oil Chemist Soc. 72, 1287 (1995)] can be used; an approach using nonenzymatic synthesis of phospholipids including phosphatidylserine in the presence of DHA-CoA has been described as well [Testet, et. al., Nonenzymatic synthesis of glycerolipids catalyzed by imidazole, J. Lipid Res. 43, 1150 (2002)]. However, these methods are expensive for large scale and/or industrial preparation.
The process for chemically synthesizing highly enriched 1,2-diDHA—containing molecular species of phosphatidylserine (PS) has been reported [Morillo, et., al., Synthesis of 1,2-diacyl-sn-glycerolphosphatidylserine from egg phosphatidylcholine by phosphoramidite methodology, Lipids 31, 541 (1996)]]. However, the safety of chemically synthesized diDHA PS has been not documented and therefore questioned. The pharmacological effect of diDHA PS species has never been reported as well.